Neuro 2 Flashcards
Describe what experimental approaches have been used for P2X receptors to determine where they are expressed
Expression cloning:
Start with tissue or cell line expressing protein of interest at high levels (P2X receptors) - rat vas deferens or PCI2 cells
Isolate mRNA
Reverse transcribe mRNA to produce cDNA molecules (more stable)
Introduce cDNA moelcules into vector
Insert vector into host cells (usuall E.coli)
Grow clones to create cDNA library
Screen library to isolate target protein cDNA
Subdivide DNA library into 10 pools
Transcribe RNA from pools and inject into Xenopus oocytes
Test oocytes for expression of protein of interest for P2X receptors-ATP mediated responses
If membrane potential changes, ATP is affecting proteins
Select positive pool and subdivide and re-test, keep selecting and subdividing until you reach a single clone which contains the cDNA of interest
DNA sequence this clone to give P2X DNA sequence
Northern analysis of P2X receptors:
mRNA extracted from tissue of interest
mRNA sample run on agarose gel to separate size
Prepare radiolabelled cDNA for sequence of interest
Probe gel and expose autoradiograph
In situ hybridisation:
Radiolabelled probes to visualise mRNA on tissue sections
Probes are anti-sense and recognise the mRNA
Gives an indication of cellular distribution
Immunohistochemical localisation:
Identify unique amino acid sequence in receptor
Immunise with peptide encoding unique sequence
Isolate antipeptide antibody
Prepare tissue sections and stain with antibody
Visualise staining with a tagged antibody
Describe what experimental approaches have been used for P2X receptors to determine their subunit stoichiometry
Western blotting:
Normally you denature and run proteins as monomers (separates by molecular weight)
- does not tell you native conformation
Chemically crosslink proteins using PPAPA and use a non-reducing gel
Find crosslinked P2X is 3x the weight on monomer - therefore P2X forms as a trimer
Describe what experimental approaches have been used for P2X receptors to determine whether they can form heteromeric channels
Some native P2X receptors don’t respond to ATP in the same way as when you just express one P2X subunit which suggests heteromeric channels can be formed.
Heteromeric channels show composite phenotypes
Patch clamp recordings from neurons and recombinant P2X receptors:
Patch clamp nodose sensory neurons and add ATP - see sustained inward current
Wash away ATP and add a-B-meATP - no change in inward current
Patch clamp recombinant P2X2 receptors and add ATP - see normal inward current
Wash of and add a-B-meATP - doesn’t evoke a current
Patch clamp recombinant P2X3 receptors and add ATP - see inward current but only transient
Wash off and add a-B-meATP - see only transient current again
P2X2 gives time course and P2X3 gives pharmacology of nodose sensory neurons
If you then measure P2X2/3 receptors that are co-expressed - you see the same response to ATP and a-B-meATP as the nodose neurons (composite phenotype)
Demonstrates that a hetermeric P2X receptor is formed in nodose neurons
What subunit combinations are possible?
Co-immunoprecipitation studies:
Tag receptors with different epitopes e.g. P2X1-FLAG, P2X2-HA
Co-express proteins in cells
Lyse cells and immunoprecipitate using anti-FLAG antibody (recognises one protein of the two) - add bead
Heavy so easy to separate - remove from beads
Run the separated anti-FLAG solution on immunoprecipitate gel and probe with anti-HA antibody
If co-assemble, will see band with anti-HA antibody
You can see which P2X subunits interact
How do you generate KO mice?
Embryonic stem cells are pluripotent
ES cells can be grown in tissue culture and so the DNA can be manipulated
To delete the gene of interest, replace that section of the DNA using a replacement vector - a piece of DNA that will remove the gene of interest and replace it with an antibiotic resistance gene
Grow ES cells and electroporate (treat with an electric shock) - this will cause the replacement vector to be taken up by some cells
By homologous recombination, replacement DNA will be taken up into some cells
Grow cells on antibiotic and only those taht have incorporated the replacement vector will grow
ES cells incorporating gene deletion can then be injected into a 16 cell stage blastocyst, this egg will then have a mixture of normal and KO cells
The blastocyst can then be injected into a pseudo-pregnat mouse and wait for pups to be born
Pups will have some tissues derived from normal and some from KO ES cells
Blastocysts from black mice, ES cells from white mice
Grey mice indicate that one parent has incorporated KO ES cells into gonads
Breed grey mice to generate KO colony
Check they are KO using molecular techniques e.g. PCR or immunohistochemistry
Study role of KO by comparing to normal mice
Describe what experimental approaches have been used for P2X receptors to determine the membrane topology of P2X receptors
Hydrophobicity analysis:
Each amino acid has a score of hydrophobicity
Calculate the mean hydrophobicity score of 10 adjacent amino acids, if hydrophobic - significant positive values, move scoring region along sequence of protein
Hydrophobicity analysis showed 2 TM domains (two hydrophobic peaks) (a-helix of 20aas would be long enough to cross the lipid bilayer)
Test model based on hydrophobicity analysis using
Glycosylation studies:
Glycosylation is addition of sugars to extracellular domains - occurs inside the endoplasmic reticulum during protein biosynthesis
P2X receptor - 3 proposed sites for N-linked glycosylation
These sites can be mutated and the effects tested
Western blot:
Run protein extracts on a gel and probe with P2X antibody with epitope tag
Higher molecular weight than predicted suggests glycosylation
Can reverse glycosylation with enzyme endoglycosidase H to see if it now = predicted molecular weight
Mutate glycosylation sites (shows P2X2 is glycosylated and glycosylated where we thought) - delta 3N P2X2 no longer glycosylated
Find boundaries of the extracellular loop:
Put mutants in where you think the boundary is located, if it increases in molecular weight it has been glycosylated so must be extracellular
Describe what experimental approaches have been used for P2X receptors to determine the contributions of the 2 transmembranes to the pore
SCAM (substituted cysteine accessibility method):
MTSEA and MTSET only bind to cysteines that are exposed on the surface of the receptor, WT receptor - no free cycteine residues so MTSEA and MTSET have no effect on P2X2 receptor properties
MTSEA is small and can permeate P2X receptors
MTSET is large and relatively impermeant
Make systematic cysteine substitution through TM2 and probe with MTSEA and MTSET
Add ATP - record voltage - see sustained inward current to application of ATP, ATP washed off and current returns to normal - reproducible response
Add ATP and co-apply MTSEA - see which mutant is affected - found at various residues (MTSEA binds to cycteine and partially blocks pore - less current), the % block varies so gives info about how narrow the pore is at different points
Blockade indicates those residues that are likely to line the pore
MTSET is too large to get past residue 336
Some effects were dependent on how you exposed the receptor to MTSEA
Some were effects were depednent on the time of exposure to MTSEA and some were dependent on the number of times the channel was activated by ATP (1st time small reduction, 2nd bigger etc)
MTSEA inhibits 333C when channel is open or closed
MTSEA inhibits 349 only when channel is open - suggests it is past gate
Results suggest the channel gate is between residue 338 and 349
SCAM with cadmium:
Cadmium binds to cysteines for longer than sodium (the major permeant ion through P2X channels) so has a blocking effect
332 and 336 always accesible to cadmium
339 and lower - cadmium cannot modify quickly when channels are closed
Therefore gate likely to be between 336 and 339
Describe what experimental approaches have been used for P2X receptors to determine the properties of the pore
I-V relationships:
Look at reversal potential of channel - indicative of cation channel
Is there selectivity amongst cations?
Change extracellular solution and see whether it has an effect on the reversal potential
If you change the EC solution for something less permeant, the I-V curve shifts to the left
If you change the EC solution for something more permeant, the I-V curve shifts to the right
Based on the amount of change of the reversal potential from that of sodium we can calculate the relative permeability of those ions throught the ion channel
When that is done, we can look at a range of organic cation reversal potentials
Organic cation ions come in a variety of sizes so we can look at whether the size of the ion has an effect on the permeability of the channel
This is the case for P2X receptors - the bigger the size, the less permeant
Can estimate narrowest region of the pore by extrapolating the line down to the x axis (~0.8nm)
Describe what experimental approaches have been used for P2X receptors to determine whether they interact with other LGICs
Record from cells co-expressing P2X and nAChR, when you add ATP and ACh, you do not see as large a response as predicted - suggests cross inhibition between P2X and nAChR
(Opening/gating of P2X receptors closes nAChR)
Immunoprecipitation with P2X antibody (will pull down associated proteins)
Describe what experimental approaches have been used for P2X receptors to determine the location of the ATP binding site
Likely that ATP binds to EC loop as it is a charged molecule so cannot cross the membrane
ATP is negatively charged so maybe there are some positively charged conserved residues on the EC loop of all P2X receptor subtypes
Point mutantions of the receptor to change conserved amino acids to see if it has any effect on the receptor sensitivity to ATP
- Inject RNA into oocytes
- Two electrode voltage clamp to measure currents
- Western blotting to measure P2X receptor expression
Compare responses to WT
Decrease in potency indicates amino acid is important
Does ATP bind intrasubunit of one P2X receptor or does it bind intersubunit at the interface of two P2X receptors? - crosslinking analysis suggest binding at interface of two P2X receptors
Can then model ATP binding using crytal structures - need to be validated by biochemical studies as crystal structure is a snapshot - can produce artifact
The residues that were predicted to be involved in ATP binding are clustered together and shows that binding site seems to be at interface
Describe how scaffolding proteins at glutamatergic synapses contribute to receptor localization and downstream signalling
NMDA receptors bind to PDZ domain of PSD-95
AMPA receptors bind to PDZ scaffold proteins GRIP and PICK-1 (AMPARs interact with PDZ4 and 5 of GRIP)
Glutamate receptor 2 is co-localised with PICK1
Regulation of PDZ interactions by phosphorylation: protein interactionos with PDZ scaffold must be regulated to allow controlled assembly and disassembly of protein complexes
Phosphorylation of GluR2 at ser880 is linked with internalisation of AMPA receptors and long term depression
Phosphorylation of AMPA receptors at ser880 causes AMPARs to favour binding to PICK1 rather than GRIP
Increased binding to PICK1 causes internalisation of AMPARs and therefore LTD
AMPA receptors also interact with TARPs such as stargazin
Stargazin is important for ensuring delivery of GluR1 to the postsynaptic site
Stargazin achors AMPA receptors in place by interaction with PSD-95
mGluR bind to the synaptic scaffolding protein Homer
Homer contains a domain with homology to EVH1 domain, can bind to other proteins
Coupling of mGluR/Homer and PSD-95 complexes by SHANK proteins
GKAP binds to guanylate kinase (GUK) domain of PSD-95
GKAP also binds SHANK
SHANK has domains which allow multimerisation, binding to Homer (linking it to GluRs), binding to actin crosslinking protein cortactin (link to cytoskeleton), PDZ bind GKAP which binds to GK domain of PSD-95 (link to NMDARs)
Coupling of mGluR/Homer and PSD-95 complexes by SHANK may cluster mGluRs
Functional role of scaffolding proteins in postsynaptic signalling and synaptic plasticity:
Coupling of NMDA receptors to NOS by PSD-95 - NOS produce NO which stimulates GC to produce cGMP which has an importnant role in synaptic plasticity
The Homer-mGluR-IP3R complex - Homer crosslinks mGlu and IP3 receptors in a functional complex. Synaptic activity upregulates expression of Homer 1a which could then bind to mGlu or IP3 receptors, since Homer 1a lacks to CC domain required for multimerisation this would uncouple mGlu and IP3 receptors. mGluR activation causes increase in cytosolic calcium concentration
The AMPAR-stargazin-PSD-95 complex - stargazin links AMPAR to PSD-95. Key in LTP - learning and memory
Some AMPARs may associate with cornichon proteins rather than TARPs. Association with CNIHs and TARPs mediate trafficking of AMPARs to the cell surface, CNIHs slow the deactivation and desensitisation of AMPARs
Describe the structure of GABA receptors. How do the actions of GABAA receptors influence neuronal activity in the central nervous system and why do their actions change during development?
GABA receptors are pentameric with 4 TM spanning domains, large EC N terminus, long IC loop between M3 and M4
There are 7 GABAA receptor subunits - subtype divergence enables receptors to have different properties
GABA receptors - agonist = GABA
Chloride is not the only permeable ion - HCO3-
If the cell is resting at -70mV and GABA is applied, Vm doesn’t change
- Chloride goes in
- HCO3 goes out - leaves behind H+ causing IC acidification
- Can affect many other cellular processes
During developmental IC chloride concentration is very variable - developing neurons = 40mM, mature neurons = 4-10mM
NKCC1 pump allows K in and brings in 2 Cl-ions in immature neurons keep [Cl-]i high
KCC2 pump is added in mature neurons gets rid of chloride ions out of the cell
Changes from inward to outward current - excitatory during development and changed to inhibitory in mature neurons
What do astrocytes do? How do astrocytes differ from oligodendrocytes and microglia?
Astrocytes are star shaped glial cells of the CNS. They regulate concentration of ions and NTs in the EC space; modulate synaptic signalling; promote formation of new synapses; supply energy to neurons; protects cells from oxidative stress.
Oligodendrocytes are also glial cells in the CNS but are involved in the formation of myelin in the CNS. These insulate axons and supply energy to axons (transport molecules from tge blood to neurons to overcome the blood-brain barrier)
Microglia are glial cells derived from mesoderm that function as macrophages (scavengers) in the central nervous system and form part of the reticuloendothelial system
Describe the synaptic and cellular mechanisms that give rise to input specific long-term potentiation in the CA1 region of the hippocampus.
Input specificity: LTP is specific to the active pathway i.e. following the rules of cooperativity and associativity (Cooperativity: LTP can be induced by either a strong tetanic stimulation to a synapse or cooperatively via the weaker stimulation of more than one pathway; Associaticity: the contributing fibres and the post synaptic cell need to be active togther - similar to classical conditioning because information from two separate sources must be coincident in both time and space)
Cellular mechanism of LTP:
Activation of Schaeffer collaterals (SCs) - release of glutamate - glutamate binds to NMDARs and AMPARs activating AMPARs but not NMDARs because NMDARs have Mg bung blocking them - activated AMPARs open mediating current flow (Na in, K out) - this may be sufficient to depolarise the cell - if enough AMPARs are activated then the depolarisation will be enough to push the Mg bung out allowing calcium in through NMDARs to cause CICR - calcium can activate calmodulin which activates CAMKII which then leads to phosphorylation of AMPAR which changes its properties (perhaps conductance) (short tem effects) - CAMKII can also lead to the insertion of more AMPARs into the membrane (longer term) - NMDAR important for induction of LTP but not maintaining the change - this is done by AMPARs
Ca/calmodulin modulation of AC leading to formation of cAMP - cAMP activates PKA which activates MAPK which translocates to the nucleus and activates CREB1 which activates CRE which transcribes growth factors to produce new spines (even longer term)
New spines have to be co-localised with PSD-95 to form proper synapse - needs consolidation
Does LTP subserve memory formation?
NMDA KO experiments:
NMDA1 KO in CA1 region of hippocampus
In CA1 - they could measure a response that was AMPA based but no response to NMDA receptor
Then showed that in CA1 NMDAR KO mice when they elicited a stimulus that would normally give LTP, they saw a transient increase but the responses came back to baseline
In the control animals, normal LTP was elicited
Behavioural test - Morrix water maze:
Mouse has to swim to platform - milk powder in water so cannot see platform - by chance reaches platform
Measure time taken to reach platform on consecutive days - should see a decrease in time need for WT mice as they should learn where the platform is due to cues around the room
Ca1-KO mice were slower at learning the hidden platform version of the morris water maze, CA1-KO mice seem normal in the landmark task water maze (shows they can perform some tasks but lose ability to form spatial memory)
Describe the general structure and properties of voltage-gated potassium channels and discuss how these properties contribute to setting resting membrane potentials and other aspects of neuronal function. How could dysfunction of these channels contribute to disease?
Potassium channels are tetrameric TM proteins with a potassium permeable pore formed by H5 and voltage activation (S4) and voltage inactivation (N and C type)
The pore is selctive for K ions and has a conserved G-Y-G sequence
Depolarisation is sensed by S4 and causes opening of K channels causing K to flood out of the cell causing repolarisation of APs - drives membrane potential towards K equilibrium potential (-90mV)
K channels reduce excitability of the cell by bring the membrane potential back to resting membrane potential
They also enable fast AP firing for example they are required in auditory processing - Kv1 and Kv3 channels are located in the MNTB. Kv1 raises threshold so that one presynaptic input = one postsynaptic output and Kv3 is activated during AP and accelerate AP repolarisation allowing cells to have shorter APs and fire at higher rates. This allows accurate timing of APs and comparison of info from both ears.
Kv1 channels are also expressed at nodes of Ranvier under the myelin sheath
Dysfunction in these channels:
Reduce the ability to reduce excitability - may result in epilepsy, fits etc?
